Estimating HIV incidence is an important challenge for public health. An assay that could reliably distinguish incident (new) HIV infections from chronic ones would be invaluable. Initial approaches to this problem have focused on the qualities of the host's humoral response to the virus. Unfortunately, assays based on this principle have been technically difficult and there are concerns about their accuracy. An alternative strategy is to measure the genetic diversity of a patient's virus, with diversity expected to be low in new HIV infections. Directly sequencing viral samples is expensive; measuring sequence diversity through high-resolution melting curves is more affordable, but it is only a qualitative method, and it may be misleading in patients with multiple founder viruses. Here, we propose to adapt a low-cost assay based on DNA hybridization kinetics, called AmpliCot, to the measurement of HIV gene complexity. The first aim of the project is to adapt and simplify the existing method (used to measure T cell receptor gene complexity) to viral envelope genes. This will require the synthesis of new measurement standards and the validation of new experimental conditions. The second aim is to determine how well AmpliCot measurements correlate with viral sequence complexity, sequence diversity, and Shannon entropy (a measure of sequence complexity weighted for relative abundance), with comparison to deep sequencing as a gold standard. The third aim is to test the predictive power of AmpliCot, high-resolution melting, or a combination of the two methods, against a test panel of plasma samples from subjects with new and chronic HIV infections. Serial samples from newly-infected HIV patients can be used to test whether the assay can detect an increase in viral genetic diversity over time. The reagents, methods and preliminary data from this pilot project will be used to support an R01 application for more extensive validation of this method.